Hematopoietic stem cell (HSC) transplantation is the most effective therapy for life-threatening hematopoietic diseases such as leukemia and many solid tumors. However such therapy is significantly limited by the shortage of HSC resources. In vitro HSC expansion is believed to be the most effective and applicable strategy to address this problem. Despite significant efforts toward this end, our ability to expand HSCs remains clinically unsuccessful because the key factors that promote HSC self-renewal have not been identified. Two reasons might explain why we failed to do so: 1) the levels of the factor(s) in bone marrow (BM) are very low which cannot be detected by previous used techniques; 2) a proper combination of factors might be required which needs a reliable assay to determine. HSC behaviors are tightly controlled by specialized BM niche cells and their secreted factors. We found that in mice, HSCs in the BM expand by approximately 100 times during the 1-2 weeks of postnatal development. We also detected an approximately 4-fold increase in functional HSC numbers on day 4 following a single treatment with 5FU. By taking the advantage of these two HSC expansion models, we examined the dynamic changes of niche cells by flow cytometry and their gene expression profiles by RNA-sequence. Such assays allow us to sensitively detect the changes in lower frequency of niche cells at 0.01% level and lower expressing genes. We specifically searched for the niche cells and their secreted factors that were increased prior to HSC expansion in both models. We speculate that such niche cells and factors might be potential candidates of HSC stimuli. In addition, we also developed a very simple and reliable ex vivo assay to evaluate the expansion of functional HSCs. We found that the dynamic changes of the CD31high endothelial cells and CD140a+CD51+ mesenchymal stem cells (MSCs), as well as a group of 78 cytokine-encoding genes expressed by these two types of niche cells are closely correlated with HSC expansion in both models. Among these cytokines, by functional analysis, we have already shown that R-spondin 2 (Rspo2) and Rspo3, Wnt agonists, can promote 15-20-fold expansion of murine HSCs in a Wnt-dependent manner. We intend to study the molecular mechanism by which Rspo2 stimulates HSC expansion (Aim 1). We will further characterize the phenotype of CD140a+CD51+ MSCs and CD31hi cells and determine the molecular mechanism by which these two types of niche cells synergistically promote HSC expansion (Aim 2). We want to determine how long the Rspo2 can sustain HSC expansion in vitro and whether Rspo2 induces genomic mutations in HSCs. We also want to evaluate whether Rspo2, CD140a+CD51+ MSCs and CD31hi cells can promote human cord blood HSCs and mobilized peripheral blood HSCs (Aim 3). We expect to identify the key stimuli of HSC self-renewal and develop an improved in vitro HSC expansion system which can be used for clinical HSC transplantation therapy. The results expected from this study will also help us to understand the molecular mechanism by which HSC activity is regulated in both normal and pathological hematopoiesis.